Dry hemostatic compositions and methods for their preparation

ABSTRACT

Dry cross-linked gelatin compositions are prepared that rapidly re-hydrate to produce gelatin hydrogels suitable as hemostatic sealants. Gelatin is cross-linked in the presence of certain re-hydration aids, such as polyethylene glycol, polyvinylprovidone, and dextran, in order to produce a dry cross-linked gelatin powder. The use of the re-hydration aids has been found to substantially increase the re-hydration rate in the presence of an aqueous re-hydration medium, typically thrombin-containing saline.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to collagen andcollagen-derived compositions and methods for their preparation. Inparticular, the present invention relates to a method for producing adry cross-linked gelatin or other collagen or collagen-derivedcomposition which is capable of absorbing water at an enhanced rate.

[0003] Fusion Medical Technologies, Inc., assignee of the presentapplication, produces a hemostatic composition under the FloSeal® tradename. The FloSeal® product is available in a package including twosyringes. A first syringe is filled with granules of cross-linked bovinegelatin which are pre-hydrated with a buffer solution. The gelatinhydrogel contains about 85% (w/w) water and is in the form of a flowablehydrogel. Immediately prior to use in the operating room, thrombin inaqueous saline is mixed with the gelatin hydrogel. The thrombin isprepared in saline and drawn up in a second syringe, and the syringesare connected together permitting mixing of thrombin and the gelatin.

[0004] The resulting mixture of the gelatin hydrogel granules and thethrombin has been found to be a highly effective hemostatic sealant whenapplied to a bleeding site. Typically, the sealant will be appliedthrough the syringe in which it has been mixed to the bleeding site.Blood will percolate through the resulting bed of hydrogel granules, andthe thrombin reacts with fibrinogen in the blood to form a fibrin clotaround the gelatin to seal the bleeding site.

[0005] Although highly effective, the present FloSeal® product has alimited shelf life. It is believed that the stability of the gelatin isreduced by hydrolysis of the packaged hydrogel. To limit possiblehydrolytic degradation, the FloSeal® product is usually shipped in atemperature-protected packaging.

[0006] For these reasons, it would be desirable to provide improvedhemostatic sealing compositions of the type which combine a collagen,gelatin, or other collagen-derived hydrogel with a thrombin-containingaqueous solution. In particular, it would be desirable to provide suchcompositions in a form which would be resistant to hydrolyticdegradation and which would therefore have a longer shelf life. It wouldbe particularly desirable to provide improved compositions having bothcomparable hemostatic activity to the present FloSeal® product andlonger shelf lives. Such compositions would be most beneficial if theycould be rapidly re-hydrated for subsequent use, typically so that theycould be extruded through a syringe. At least some of these objectiveswill be met by the inventions described hereinafter.

[0007] 2. Description of the Background Art

[0008] The FloSeal® product available from Fusion Medical Technologies,Inc., is described in Hood et al., Efficacy of Topical Hemostat FloSeal™in Vascular Surgery, an Abstract funded by Fusion Medical Technologies,Inc., which was publicly presented in September 1999. Patents coveringthe FloSeal® product include U.S. Pat. Nos. 6,063,061 and 6,066,325. Adual syringe system suitable for mixing and delivering a collagen,gelatin, or other collagen-derived component and a thrombin component ofthe FloSeal™ product is described in U.S. Pat. No. 5,908,054. Thecomplete disclosures of each of these patent references is herebyincorporated by reference.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides improved hemostatic sealingcompositions, methods for preparing such improved compositions, and kitscomprising the improved compositions. The methods and compositions willbe particularly useful for providing hemostasis at bleeding sites,including surgical bleeding sites, traumatic bleeding sites and thelike. An exemplary use of the compositions may be in sealing the tissuetract above a blood vessel penetration created for vascularcatheterization.

[0010] The compositions comprise a dry cross-linked gelatin powder whichhas been prepared to re-hydrate rapidly. The gelatin powder preferablycomprises relatively large particles, also referred to as fragments orsub-units, as described in U.S. Pat. Nos. 6,063,061 and 6,066,325, thefull disclosures of which have previously been incorporated byreference. A preferred particle size will be the range from 150 μm to750 μm, but particle sizes outside of this preferred range may find usein many circumstances. The dry compositions will also display asignificant “equilibrium swell” when exposed to an aqueous re-hydratingmedium. Preferably, the swell will be in the range from 400% to 1000%,but may fall outside of this range as set forth in the above-referencedpatents. “Equilibrium swell” may be determined by subtracting the dryweight of the gelatin hydrogel powder from its weight when fullyhydrated and thus fully swelled. The difference is then divided by thedry weight and multiplied by 100 to give the measure of swelling. Thedry weight should be measured after exposure of the material to anelevated temperature for a time sufficient to remove substantially allresidual moisture, e.g., two hours at 120° C. The equilibrium hydrationof the material can be achieved by immersing the dry material in asuitable re-hydrating medium, such as aqueous saline, for a time periodsufficient for the water content to become constant, typically for from18 to 24 hours at room temperature.

[0011] The dry cross-linked gelatin powders of present invention willusually have some residual moisture, but will be sufficiently dry toachieve the desired stability and extended shelf life. Typically, thedry compositions will have a moisture content below 20% by weight (w/w)or less, preferably having a moisture content in the range from 5% byweight to 15% by weight. To maintain dryness, the compositions willtypically be packaged in a manner suitable to prevent moistureincursion, as described in more detail in connection with the kits ofthe present invention.

[0012] In one particular aspect of the present invention, compositionswill comprise cross-linked gelatin powders having a moisture content of20% (w/w) or less, wherein the powder was cross-linked in the presenceof a re-hydration aid so that the powder has an aqueous re-hydrationrate which is at least 5% higher than the re-hydration rate of a similarpowder prepared without the re-hydration aid. The “re-hydration rate” isdefined herein to mean the quantity of an aqueous solution, typically0.9% (w/w) saline, that is absorbed by a gram of the powder (dry weightbasis) within thirty seconds, expressed as gm/gm. Particular techniquesfor measuring this rate are described in the Experimental sectionhereinafter. Preferred compositions of the present invention will have are-hydration rate of at least 3 gm/gm, preferably at least 3.5 gm/gm,and often 3.75 gm/gm or higher. Re-hydration rates of similar powdersprepared without the re-hydration aids are typically below three, and apercentage increase in re-hydration rate will usually be at least 5%,preferably being at least 10%, and more preferably being at least 25% orhigher.

[0013] The dry cross-linked gelatin powders of the present inventionhaving improved re-hydration rates are preferably obtained by preparingthe powders in the presence of certain re-hydration aids. Suchre-hydration aids will be present during the preparation of the powders,but will usually be removed from the final products. For example,re-hydration aids which are present at about 20% of the total solidscontent, will typically be reduced to below 1% in the final product,often below 0.5% by weight. Exemplary re-hydration aids includepolyethylene glycol (PEG), preferably having a molecular weight of about1000; polyvinylpyrrolidone (PVP), preferably having an average molecularweight of about 50,000; and dextran, typically having an averagemolecular weight of about 40,000. It is preferred to employ at least twoof these re-hydration aids when preparing the compositions of thepresent invention, and more particularly preferred to employ all three.

[0014] The methods of the present invention thus comprise providing anaqueous solution of a non-cross-linked gelatin combined with are-hydration aid. The non-cross-linked gelatin will typically be presentin an aqueous solution at from 5% (w/w) to 15% (w/w) and there-hydration aids will be typically present from 5% to 30% (w/w) basedon the weight of gelatin in the aqueous solution. Preferably, there-hydration aid comprises PEG at from 2.5% to 20% (w/w) based on theweight of the gelatin, PVP at from 1.25% to 20% (w/w), and dextran atfrom 1.25% to 20% (w/w).

[0015] The non-cross-linked gelatin together with the re-hydration aidis then cross-linked in any manner suitable to form the hydrogel. Forexample, polymeric molecules may be cross-linked using bi- orpoly-functional cross-linking agents which covalently attach to two ormore polymer molecules chains. Exemplary bifunctional cross-linkingagents include aldehydes, epoxies, succinimides, carbodiimides,maleimides, azides, carbonates, isocyanates, divinyl sulfone, alcohols,amines, imidates, anhydrides, halides, silanes, diazoacetate,aziridines, and the like. Alternatively, cross-linking may be achievedby using oxidizers and other agents, such as periodates, which activateside-chains or moieties on the polymer so that they may react with otherside-chains or moieties to form the cross-linking bonds. An additionalmethod of cross-linking comprises exposing the polymers to radiation,such as gamma radiation, to activate the polymer chains to permitcross-linking reactions. Dehydrothermal cross-linking methods may alsobe suitable. Preferred methods for cross-linking gelatin molecules aredescribed below.

[0016] Exemplary methods for producing cross-linked gelatins are asfollows. Gelatin is obtained and suspended in an aqueous solution toform a non-cross-linked hydrogel, typically having a solids content from1% to 70% by weight, usually from 3% to 10% by weight. The gelatin iscross-linked, typically by exposure to either glutaraldehyde (e.g.,0.01% to 0.05% w/w, overnight at 0 C. to 15 C. in aqueous buffer),sodium periodate (e.g., 0.05 M, held at 0° C. to 15° C. for 48 hours) or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (“EDC”) (e.g., 0.5% to1.5% w/w overnight at room temperature), or by exposure to about 0.3 to3 megarads of gamma or electron beam radiation. Alternatively, gelatinparticles can be suspended in an alcohol, preferably methyl alcohol orethyl alcohol, at a solids content of 1% to 70% by weight, usually 3% to10% by weight, and cross-linked by exposure to a cross-linking agent,typically glutaraldehyde (e.g., 0.01% to 0.1% w/w, overnight at roomtemperature). In the case of aldehydes, the pH should be held from about6 to 11, preferably from 7 to 10. When cross-linking withglutaraldehyde, the cross-links are formed via Schiff bases which may bestabilized by subsequent reduction, e.g., by treatment with sodiumborohydride. After cross-linking, the resulting granules may be washedin water and optionally rinsed in an alcohol, and dried. The resultingdry powders may then be loaded into the applicators of the presentinvention, as described in more detail hereinafter.

[0017] After cross-linking, at least 50% (w/w) of the re-hydration aidwill be removed from the resulting hydrogel. Usually, the re-hydrationaid is removed by filtration of the hydrogel followed by washing of theresulting filter cake. Such filtration/washing steps can be repeated oneor more additional times in order to clean the product to a desiredlevel and to remove at least 50% of the re-hydration aid, preferablyremoving at least 90% (w/w) of the re-hydration aid originally present.

[0018] After filtration, the gelatin is dried, typically by drying thefinal filter cake which was produced. The dried filter cake may then bebroken up or ground to produce the cross-linked powder having a particlesize in the desired ranges set forth above.

[0019] Kits according to the present invention will comprise a firstcontainer holding the dry cross-linked gelatin powder of the presentinvention, as described above. The kits will further comprise a secondcontainer holding an aqueous re-hydration medium, typically a saline orother aqueous solution comprising thrombin which is intended to be mixedwith the gelatin as the gelatin is re-hydrated. The containers can be inany form, but will preferably be in the form of syringes which permitmixing of the dry gelatin with the re-hydration medium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 illustrates a kit constructed in accordance with theprinciples of the present invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0021] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLES 1 Preparation of Gelatin Powder

[0022] Strips of bovine corium were suspended in a sodium hydroxidesolution of concentration 1 M to 2 M for 1 hr at room temperature,neutralized with phosphoric acid, and rinsed. The treated strips werethen resuspended in deionized water, adjusted to pH 7-8, and heated to70° C. A homogenizer was used to further reduce the size of the strips.After 1 hr at 70° C., the corium was largely solubilized to gelatin. Theamount of corium was chosen so that the solids content of the resultinggelatin solution was approximately 3-10% (w/w), typically 7-10%. Thesolution was cast as thin layers onto Teflon® coated metal trays, dried,and ground to form gelatin powder.

EXAMPLE 2 Preparation of “Modified Gelatin Powder”

[0023] Re-hydration aids (Table 1) were dissolved in 500 mL of 50° C.de-ionized water and then an amount of bovine derived gelatin powder,prepared as in Example 1, was added to the solution. The finalconcentration of gelatin in solution was chosen to be approximately 8%(w/w, bulk gelatin powder basis), and the total amount of re-hydrationaids in the solution was chosen as in Examples 9-44 (Tables 1 and 2).After the gelatin had dissolved, the solution was poured into Teflon®coated metal trays and dried. The dried gelatin sheet is ground to form“modified gelatin powder”.

[0024] Alternatively, strips of bovine corium were suspended in a sodiumhydroxide solution of concentration 1 M to 2 M for 1 hr at roomtemperature, neutralized with phosphoric acid, and rinsed. The treatedstrips were then resuspended in deionized water, adjusted to pH 7-8, andheated to 70° C. A homogenizer was used to further reduce the size ofthe strips. After 1 hr at 70° C., the corium was largely solubilized togelatin. The amount of corium was chosen so that the solids content ofthe resulting gelatin solution was approximately 3-10% (w/w), typically7-10%. Amounts of re-hydration aids were chosen as in Examples 9-44(Tables 1 and 2) and were then added to the gelatin solution, either insolid form or dissolved in a small volume of water. The solution wascast into thin layers onto Teflon® coated metal trays, dried, and groundto form “modified gelatin powder”. Examples of several formulations formodified gelatin are given in Tables 1 and 2.

EXAMPLE 3 Preparation of Cross-linked Gelatin Powder from “ModifiedGelatin powder”

[0025] 600 mL of 0.2 M phosphate buffer (pH 9.2±0.2) was cooled to atemperature below 12° C. 0.32 mL of glutaraldehyde (25%) was added tothe buffer solution and then 20 g of modified gelatin powder was added,resulting in a glutaraldehyde concentration of 4000 ppm (glutaraldehydeto modified gelatin, bulk weight basis). The gelatin was suspended inthe glutaraldehyde solution with a stir bar. The pH of each suspensionswas adjusted to a range of 9.2±0.2 and then maintained at a temperatureof 9 to 12° C. and pH of 9.2±0.2 over 19 hours.

[0026] The suspension was filtered and the filter cake was washed withde-ionized water three times by completely covering the filter cake withde-ionized water and then allowing the vacuum to draw the rinse waterthrough the cake. The filter cake was left in the funnel during eachrinse.

[0027] 0.2 g of NaBH4 was dissolved in 600 mL 25 mM phosphate buffer, pH7.4 0.2, in a beaker. The above filter cake was suspended in the NaBH4solution at room temperature (about 22° C.) for 3 hours, then filteredto remove the liquid.

[0028] The filter cake was next suspended in 600 mL of buffer solutionat room temperature (about 22° C.) for 30 minutes and filtered again.The buffer was composed of sodium phosphate (dibasic anhydrous andmonobasic monohydrate) and sodium ascorbate. The above procedure wasrepeated twice to ensure that the appropriate ratio of salts to gelatinwere present to form the desired buffer composition upon reconstitution.The filter cake was dried, then ground with a Waring Blender, resultingin “cross-linked gelatin powder”.

[0029] This method was also used to prepare cross-linked gelatin powderfrom unmodified gelatin powder; that is, gelatin to which nore-hydration aids were added during its preparation.

EXAMPLE 4 Preparation of Irradiated Product from Cross-linked GelatinPowder

[0030] About 800 mg (bulk weight) of the cross-linked gelatin powder,prepared as in Example 2, were put into each of several 5 cc syringes.The syringes containing powder were sterilized with gamma irradiation atambient temperature.

EXAMPLE 5 Use of Product as a Hemostatic Agent

[0031] A syringe of product containing approximately 0.8 g of irradiatedcross-linked gelatin powder was prepared from modified gelatin powder.The modified gelatin powder was prepared as in Example 2. The modifiedgelatin was further cross-linked and irradiated as in Examples 3 and 4.The product was mixed with 4 mL of a saline solution containing about1000 Units of bovine thrombin per milliliter. Mixing was achieved bypassage back and forth between two syringes connected with afemale-female straight-through Luer connector. The powder in the syringewas hydrated as it mixed with the thrombin solution, forming granules ofhydrogel.

[0032] A square lesion, approximately 1 cm×1 cm×0.2 cm deep, was createdon the liver of a farm-grade pig. The pig had been anticoagulated withheparin so that its activated clotting time (ACT) was three to fivetimes its baseline value, and the lesion bled freely prior to treatment.After about 30 seconds from the start of mixing, approximately 2 mL ofthe hydrated powder was extruded from the syringe onto the lesion andheld in place with compression for two minutes. After compression wasremoved, the treated lesion was observed for bleeding at 3 min, 10 min,and 50 min after application. No bleeding was seen from the treatedlesion at the 3 min and 10 min observation. After the 10 minobservation, the treated lesion was irrigated with saline solution.While excess material was removed, no re-bleeding was observed. At 50min after application, the lesion was observed again and no bleeding wasseen.

EXAMPLE 6 Determination of Re-hydration Rate of a Powder

[0033] The “re-hydration rate” of a powder was measured as follows. Thepowder, packed in a 5 cc syringe, was mixed with a syringe containing avolume of aqueous solution by passage between the two syringes connectedwith a Luer fitting for 30 seconds. The volume of aqueous solution waschosen to be in excess of what could be expected to be absorbed in 30seconds. Typically, 0.8 g (bulk weight) of powder was mixed with 3 mL of0.9% sodium chloride solution. The resulting mixture was thenimmediately filtered to remove any unabsorbed liquid. The wet filteredmaterial was weighed, then dried in a 120° C. oven for two hours andre-weighed. This measurement gave the total amount of water removed fromthe wet material and the weight of the dry powder. The amount of waterthat had been absorbed by the powder was then calculated after a smallcorrection is made for the residual moisture that had been present inthe powder originally. The “re-hydration rate” was given as the mass ofsaline solution absorbed per gram dry weight of powder in that 30 secondinterval.

[0034] In the calculation below, the fraction solids of the bulk powder(“S”) was measured independently by drying the bulk powder at 120° C.for 2 hr and weighing the powder before and after drying. The value of Sis given by the following:$S = \frac{{w\quad e\quad i\quad g\quad h\quad t\quad a\quad f\quad t\quad e\quad r\quad d\quad r\quad y\quad i\quad n\quad g{\quad \quad}a\quad t\quad 120{^\circ}\quad {C.}},{2\quad h\quad r}}{w\quad e\quad i\quad g\quad h\quad t\quad b\quad e\quad f\quad o\quad r\quad e\quad d\quad r\quad y\quad i\quad n\quad g}$

[0035] Re-hydration rate calculation:

[0036] A: initial weight of the pan and filter paper

[0037] B: weight of the pan, filter paper and hydrated powder

[0038] C: weight of the pan, filter paper and sample after drying inoven

[0039] S: fraction solids of the bulk powder originally in syringe

[0040] M: grams of saline absorbed per gram of powder (dry weight)during mixing (“absorption rate”)$M = \frac{( {B - A} ) - {( {C - A} )/S}}{( {C - A} )}$

EXAMPLE 7 Re-hydration Rate and Physical Property Determination forSeveral Batches of Powder Product

[0041] Tables 1 and 2 depict the results of re-hydration ratemeasurements performed on one to for several batches of powder product(Examples 9-23). These were made using methods as per Examples 1, 2, 3,and 4. Except for Examples 9 and 17, these were prepared from modifiedgelatins that were made with various proportions of gelatin and thefollowing re-hydration aids: polyethylene glycol (PEG), averagemolecular weight 1000; polyvinylpyrrolidone (PVP), “k-30” designation,corresponding to an average molecular weight of about 50,000; anddextran, average molecular weight 40,000.

[0042] It is seen that use of several different combinations of gelatinand re-hydration aids can result in a powder product that absorbs moreaqueous saline solution in 30 seconds per gram of powder than powderproduct made from gelatin to which no re-hydration aids have been added.It is also seen that the combination of gelatin, PEG, PVP and dextran ata bulk weight ratio of 80:10:5:5 in the modified gelatin (Example 10)produces a powder product that absorbs about 33% more saline solutionper gram in 30 seconds than powder product made from unmodified gelatin.

[0043] Table 1 also gives values for other physical propertiesdetermined for the powder product lots. “Percent solids” was determinedby weighing the powder before and after drying at 120° C. for two hoursto drive off residual moisture. “DSC peak temperature” refers to thetemperature at which a peak is exhibited in a thermogram of adifferential scanning calorimetry measurement conducted from 1° C. to70° C. “Equilibrium swell” was determined by suspending the powder in anexcess of saline solution for at least 18 hr at room temperature. Thehydrated powder was weighed to determine its “equilibrium wet weight”and dried at 120° C. for two hours and re-weighed to determine its “dryweight”. Equilibrium swell is given as${{Equilibrium}\quad {swell}\quad (\%)} = {100\% \times \frac{{{equilibrium}\quad {wet}\quad {weight}} - {{dry}\quad {weight}}}{{dry}\quad {weight}}}$

[0044] Values for “mean particle size” were measured by light scatteringin a Coulter LS particle size analyzer.

[0045] From the data presented in Table 1, it appears that theappropriate use of re-hydration aids can change the re-hydration rate ofthe powder product without significantly changing other physicalproperties.

EXAMPLE 8 Measurement of Polyethylene Glycol, Polyvinylpyrrolidone, andDextran Levels in Modified Gelatin Powder and in Cross-linked Powder

[0046] Approximately 50 mg modified gelatin or 250 mg cross-linkedirradiated powder product were suspended in 10 mL of deionized water andheated for 3 hr at 65° C. The samples were then centrifuged at 15minutes at 2000 rpm. The resulting supernatant was filtered through a0.45 μm Gelman Acrodisc filter, the first mL being discarded. Theresulting sample was then assayed by three different high performanceliquid chromatography (HPLC) methods to quantitate the polyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), and dextran in the sample. ForPEG, 100 μL of the sample was injected onto a Waters Ultrahydrogel 120column, 7.8×300 mm, with guard column and prefilter, using deionizedwater as the mobile phase. A refractive index detector was used tomonitor the effluent. For PVP, 100 μL of the sample was injected onto aPhenomenex Kingsorb C18 5 μm column, 4.6×150 mm, with guard column andprefilter, using a gradient of methanol and aqueous sodium phosphate asthe mobile phase. An ultraviolet absorbance detector was used to monitorthe effluent. For dextran, 100 μL of the sample was injected onto aWaters Ultrahydrogel Linear column, 7.8×300 mm, with guard column andprefilter, using 0.1 M sodium phosphate, pH 7 and acetonitrile at a90:10 ratio as the mobile phase. A refractive index detector was used tomonitor the effluent. All columns were heated to 40° C. for theanalyses. The limit of quantitation was about 0.1% (w/w sample) for PEGand PVP, 0.2% (w/w sample) for dextran.

[0047] Modified gelatin was prepared as per Example 2. The modifiedgelatin was analyzed for PEG, PVP and dextran in the manner describedabove. Results indicated that PEG, PVP, and dextran were present at 16%,8%, and 3% (w/w bulk) respectively. The modified gelatin wassubsequently subjected to cross-linking, sodium borohydride treatment,and rinsing as per Example 3 to form cross-linked modified gelatinpowder. When this powder was analyzed for PEG, PVP, and dextran by HPLCin the manner described above, the content of each of the threere-hydration aids was found to be below the limit of quantitation.

EXAMPLE 9 Powder Product Made without Re-hydration Aids

[0048] Unmodified gelatin—that is, gelatin to which processing aids werenot added—was prepared from bovine corium strips as in Example 1 andcross-linked as in Example 3. The cross-linked unmodified gelatin wasthen packed into syringes and gamma irradiated as in Example 4. Physicalproperties of the resulting product were measured as in Examples 6 and 7and are given in Table 1.

EXAMPLES 10-23 Powder Product made with Re-hydration Aids

[0049] Batches of modified gelatin were prepared as in Example 2 fromgelatin powder or corium strips and from one, two, or three re-hydrationaids. Table 1 gives the proportions of bulk gelatin and re-hydrationaids used. The modified gelatin was then cross-linked as in Example 3.Except for Example 17, the re-hydration aids used were from thefollowing list: polyethylene glycol (PEG) of an average molecular weightof about 1000; polyvinylpyrrolidone (PVP), “k-30” designation, of anaverage molecular weight of about 50,000; and dextran, of an averagemolecular weight of about 40,000. In Example 17, PEG of an averagemolecular weight of about 400 was used. The cross-linked modifiedgelatin was then packed into syringes and gamma irradiated as in Example4. Physical properties of the resulting powder product from each ofthese preparations were measured as in Examples 6 and 7 and are given inTable 1. Data given with the formulation for Example 10 is the averageand standard deviation of nine batches prepared according to thatformulation.

EXAMPLES 24-44 Powder Product made with Various Re-hydration Aids

[0050] Batches of modified gelatin were prepared as in Example 2 fromgelatin powder or corium strips and from one of several re-hydrationaids. Table 2 gives the identity and concentration of re-hydration aidused in each batch as a ratio of bulk gelatin weight to re-hydration aidand as a percentage of total bulk solute used to prepare the modifiedgelatin. The modified gelatin was then cross-linked as in Example 3. Thecross-linked modified gelatin was then packed into syringes and gammairradiated as in Example 4. Physical properties of the resulting powderproduct from each of these preparations were measured as in Examples 6and 7 and are given in Table 2. Data for the Example 9 formulation isprovided in Table 2 for comparison. TABLE 1 Properties of powder productafter Target bulk weight percent in cross-linking and gamma irradiationmodified gelatin (re-hydration aids largely removed) Gelatin PEG PVPDextran DSC Mean (bulk MW = MW˜ MW = peak temp Equilibrium particle Re-Lot weight) 1000 D 50000 D 40000 D % solids (° C.) swell (%) size (μm)hydration* No re-hydration aids added Example 9 208-32 100 0 0 0 88.641.3 551 440 2.85 Preferred composition (four-way mixture) Example 10avg of 9 lots 80 10  5 5 87.6 42.1 595 423 3.79 std. deviation of 9 lots1.0 1.4 43 65 0.15 Three-way mixtures Example 11 228-69-1 80 10  10 088.1 40.8 667 387 3.51 Example 12 228-69-2 80 10  0 10 88.4 40.6 670 3673.14 Example 13 228-78 80 0 10 10 86.7 41.1 632 414 3.20 Gelatin-PEGmixtures Example 14 212-39-2 94 6 0 0 86.2 44.4 502 372 2.68 Example 15228-42-3 89 11  0 0 88.6 42.8 594 428 3.16 Example 16 228-42-1 80 20  00 88.9 42.4 575 312 3.47 Example 17 214-62-1 89  11** 0 0 87.1 40.7 599406 3.11 Gelatin-PVP mixtures Example 18 228-38-3 94 0 6 0 88.2 42.2 567399 3.26 Example 19 228-38-2 89 0 11 0 88.3 41.0 605 422 3.44 Example 20228-38-1 80 0 20 0 88.6 42.4 596 401 3.52 Gelatin-dextran mixturesExample 21 228-35-3 94 0 0 6 88.1 40.5 631 395 3.18 Example 22 228-35-289 0 0 11 88.3 41.4 598 345 3.03 Example 23 228-35-1 80 0 0 20 88.5 41.9624 392 3.01

[0051] TABLE 2 Re-hydration aid Conc'n of processing Physical propertiesaid in DSC MW or bulk modified peak Mean Re- other gelatin gelatin %temp Equilibrium particle hydration Lot type designation wt: aid (bulkwt %) solids (° C.) swell (%) size (μm) rate* Example 9 208-32 nore-hydration aids 88.6 41.3 551 440 2.85 Example 24 214-11-1 glyceroln/a 4 20% 85.5 43.4 483 653 2.19 Example 25 214-11-2 glycerol n/a 8 11%86.4 43.4 529 421 2.62 Example 26 214-11-3 glycerol n/a 16 6% 86.5 43.0543 398 2.35 Example 27 214-44-1 dextran 148000 D 4 20% 85.5 nr 634 4332.62 Example 28 214-44-2 dextran 148000 D 8 11% 85.4 nr 607 453 2.57Example 29 214-44-3 dextran 148000 D 16 6% 85.5 nr 603 527 2.33 Example30 214-44-4 dextran 148000 D 32 3% 85.7 nr 531 491 2.37 Example 31228-35-4 dextran  40000 D 32 3% 84.5 41.4 633 380 2.59 Example 32214-50-1 PVP k-90 4 20% 85.3 44.0 612 664 2.41 Example 33 214-50-2 PVPk-90 8 11% 85.6 44.3 538 581 2.71 Example 34 214-50-3 PVP k-90 16 6%85.6 44.1 527 593 2.78 Example 35 214-50-4 PVP k-90 32 3% 86.1 43.0 597538 2.76 Example 36 214-53-4 PVP k-30 32 3% 87.3 41.1 580 447 2.72Example 37 214-59-1 PEG  400 4 20% 86.7 42.0 595 407 2.18 Example 38214-66-1 PEG  400 6 14% 86.5 40.8 603 501 2.63 Example 39 212-39-1 PEG 400 16 6% 86.2 43.8 513 403 2.11 Example 40 212-39-2 PEG 1000 16 6%86.2 44.4 502 372 2.68 Example 41 214-59-3 PEG 8000 4 20% 87.4 41.5 548429 2.87 Example 42 214-66-3 PEG 8000 6 14% 86.9 41.4 581 426 3.80Example 43 214-62-3 PEG 8000 8 11% 86.8 42.0 631 511 2.78 Example 44212-39-3 PEG 8000 16 6% 86.4 44.6 546 518 2.72

[0052] While the above is a complete description of the preferredembodiments of the invention, various alternatives, modifications, andequivalents may be used. Therefore, the above description should not betaken as limiting the scope of the invention which is defined by theappended claims.

What is claimed is:
 1. A method for preparing a substantially drycross-linked gelatin powder, said method comprising: providing anaqueous solution comprising linked gelatin combined with at least onere-hydration aid; drying the solution of the gelatin and re-hydrationaid to produce solids; grinding the solids to produce a powder;cross-linking the powder; removing at least 50% (w/w) of there-hydration aid; and drying the cross-linked gelatin to produce apowder having a moisture content below 20% (w/w).
 2. A method as inclaim 1, wherein the re-hydration aid comprises at least one materialselected from the group consisting of polyethylene glycol (PEG),polyvinylpyrrolidone (PVP), and dextran.
 3. A method as in claim 2,wherein the re-hydration aid comprises at least two of the materials. 4.A method as in claim 3, wherein the re-hydration aid comprises all thematerials.
 5. A method as in claim 1, wherein the re-hydration aid ispresent at a concentration in the range from 5% to 30% by weight basedon the weight of gelatin present in the aqueous solution.
 6. A method asin claim 5, wherein the re-hydration aid comprises PEG at from 2.5% to20% by weight, PVP at from 1.25% to 20% by weight, and dextran at from1.25% to 20% by weight.
 7. A method as in claim 1 wherein cross-linkingcomprises adding a cross-linking agent to the gelatin solution.
 8. Amethod as in claim 7, wherein the cross-linking agent comprisesglutaraldehyde.
 9. A method as in claim 1, wherein removing at least 50%of the re-hydration aid comprises filtering the suspension ofcross-linked powder in a solvent followed by filtration gelatin toproduce a filter cake and washing the filter cake to remove there-hydration aid.
 10. A method as in claim 9, wherein washing the filtercake removes at least 90% (w/w) of the re-hydration aid originallypresent in the gelatin.
 11. A method as in claim 1, wherein dryingcomprises drying the filter cake after washing, wherein the methodfurther comprises drying the ground filter cake to produce the dryground gelatin powder.
 12. A composition comprising cross-linked gelatinpowder having a moisture content of 20% (w/w) or less, wherein thepowder was cross-linked in the presence of a re-hydration aid so thatthe powder has an aqueous re-hydration rate which is at least 5% higherthan the re-hydration rate of a similar powder prepared without there-hydration aid.
 13. A composition as in claim 12, wherein the powderhas a re-hydration rate which is at least 10% higher than there-hydration rate of a similar powder prepared without the re-hydrationaid.
 14. A composition as in claim 13, wherein the powder has are-hydration rate which is at least 25% higher than the re-hydrationrate of a similar powder prepared without the re-hydration aid.
 15. Acomposition as in claim 12, wherein the re-hydration aid comprises atleast one material selected from the group consisting of polyethyleneglycol (PEG) polyvinylprovidone (PVP), and dextran.
 16. A composition asin claim 15, wherein the re-hydration aid comprises at least two of thematerials.
 17. A composition as in claim 16, wherein the re-hydrationaid comprises all the materials.
 18. A composition as in claim 12,wherein the powder has a mean particle size in the range from 150 μm to750 μm.
 19. A composition as in claim 18, wherein the powder has anequilibrium swell in the range from 400% to 1000%.
 20. A kit comprising:a first container holding cross-linked gelatin powder any of claims12-19; and a second container holding an aqueous re-hydration medium.21. A kit as in claim 20, wherein the first container is a syringe. 22.A kit as in claim 21, wherein the second container is a syringe.
 23. Akit as in claim 20, wherein the aqueous re-hydration medium comprisesthrombin.
 24. A kit as in claim 20, further comprising instructions foruse setting forth a method for combining the cross-linked gelationpowder and the re-hydration medium to produce a thrombin-containingfragmented gelatin hydrogel, and applying the hydrogel to a wound site.25. A kit as in claim 20, further comprising a package holding the firstand second containers.